【摘要】 目的探讨微小RNA-7(microRNA-7,miRNA-7)对中波紫外线(UVB)照射诱导的皮肤氧化应激损伤的保护性作用及其作用机制。方法建立UVB照射诱导的人永生化表皮角质形成细胞(HaCaT)氧化应激损伤模型,脂质体转染法过表达miR-7,并设立阴性对照组(miR-NC)和空白对照组。CKK-8法测定细胞活性,流式细胞术检测细胞凋亡,比色法检测细胞中超氧化物歧化酶(SOD)及谷胱甘肽过氧化物酶(GSH-Px)活性,荧光法检测细胞内活性氧自由基(ROS)的含量,Real-time PCR和Western blot检测细胞中miR-7、Keap1及Nrf2 mRNA表达。结果与空白对照组相比,UVB模型组HaCaT细胞活性、SOD和GSH-Px活性显著下降(P均<0.05),ROS含量显著上升(P<0.05)。miR-7过表达能显著增加HaCaT细胞活性、SOD和GSH-Px活性,同时抑制Keap1蛋白的表达,增加Nrf2蛋白表达(P均<0.05),siRNA-Nrf2转染后能显著抑制Nrf2表达(P<0.05),进而逆转miR-7对细胞的保护作用。结论 miR-7能削弱UVB作用下HaCaT细胞的氧化应激损伤,其机制可能与Keap1-Nrf2信号通路的活化有关。更多还原

【Abstract】 Objective To investigate the cytoprotective effects of miRNA-7 on HaCaT cells that induced by ultraviolet irradiation B(UVB). Methods Following constructing oxidative stress injury model of HaCaT cells by exposuring to UVB,the culture cells were treated with microRNA-7 mimics and negative control(NC) was established.Then,CKK-8 assay was used to assess the proliferation of HaCaT cells,and cell apoptosis rate was analyzed by flow cytometry assay. The activities of superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px) were determined by colorimetry. Concentration of intra-cellular reactive oxygen species(ROS) was assessed by fluorescence spectrometry. Furthermore,the expression of miR-7,Keap1 and Nrf2 were determined by Real-time quantitative PCR and Western blot. Results UVB irradiation significantly reduced the HaCaT cells, proliferation,SOD and GSH-Px activity,while up-regulated the concentration of ROS(P<0.05). MiR-7 overexpression significantly increased the proliferation of HaCaT cells,promoted the activities of SOD and GSH-Px(P<0.05),as well as decreased the ROS level. Further mechanism analysis showed that miR-7 overexpression markedly inhibited Keap1 expression and increased Nrf2 expression. Moreover,Nrf2 siRNA pretreatment significantly attenuated the protecting effects brought by miR-7 overexpression. The expression of Nrf2 was marked reduced after siRNA-Nrf2 transfection and reversed the protection of miR-7 for HaCaT cell. Conclusion MiR-7 can reduce the UVB induced oxidative stress injury of HaCaT cells by regulating the Keap1-Nrf2 signal pathway.更多还原